云南省恶性疟原虫氯喹及青蒿素抗性相关基因的联合突变分析

发布时间：2018-01-13 17:26

本文关键词：云南省恶性疟原虫氯喹及青蒿素抗性相关基因的联合突变分析　出处：《中国寄生虫学与寄生虫病杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的分析云南省恶性疟原虫抗氯喹转运蛋白(Plasmodium falciparum chloroquine resistant transporter,Pfcrt)基因和青蒿素抗性相关(K13)基因的联合突变特性。方法收集2012年8月-2015年12月寄生虫病防治信息管理系统登记和报告的云南省恶性疟病例滤纸血样和流行病学史等相关信息,提取疟原虫DNA,巢式PCR扩增恶性疟原虫Pfcrt基因exon2区和K13基因kelch结构域并测序,测序序列与2655199、PF3D7_1343700参比序列进行比对,用MEGA 5.04、Arlequin 3.01软件分析Pfcrt基因exon2区、K13基因kelch结构域的单倍型、单倍型间期望杂合度(He)及其群体间的遗传分化指数(Fst)等,用Network 4.6.0软件制作单倍型中介网络进化图,采用Epi Data 3.1软件建立数据库,SPSS 21.0统计学软件进行数据分析。结果共收集恶性疟病例血样234份,Pfcrt基因exon2区、K13基因kelch结构域巢式PCR扩增产物同时成功测序192份。其中,云南省本地感染病例血样12份,自非洲、缅甸感染的病例血样分别为30和150份。218份血样的Pfcrt基因exon2区DNA序列有7种单倍型,He为0.238 5,错义突变序列占83.0%(181/218),72~76位点呈单重突变(CVMNT)、双重突变(SVMNT)、三重突变(CVIET)的比例分别为1.8%(4/218)、6.4%(14/218)和74.8%(163/218),7种单倍型以氯喹敏感型CVMNK为起始,再按单重、双重及三重突变的路径逐步进化。192份血样的K13基因kelch结构域DNA序列有21种单倍型,He为0.044 9,错义突变序列占35.9%(69/192),在16、446、676等9个位点均只发生单重突变,F446I的突变率最高,为25.0%(48/192),21种单倍型的中介网络图显示"星状"布局。云南省本地恶性疟原虫种群与缅甸种群间的Fst最小,为0.020 3。Pfcrt基因的氯喹敏感型CVMNK血样中,K13基因kelch结构域位点突变的比例为20.6%(7/34),氯喹抗性型CVMNT、CVIET血样中的检出比例分别为1/4和36.4%(51/140)。结论云南省疫情报告病例中恶性疟原虫的氯喹、青蒿素相关抗性基因的联合突变率为27.1%,且青蒿素抗性基因突变型之间可能存在种群扩张模式。
[Abstract]:Analysis of Plasmodium falciparum in Yunnan province chloroquine resistance transporter (Plasmodium falciparum chloroquine resistant to transporter Pfcrt) and artemisinin resistance related gene (K13) mutation characteristic gene. Methods from August 2012 December -2015 parasitic disease information management system for registration and report a case of falciparum malaria in Yunnan province. Blood samples and epidemiological history and other related information extraction. Plasmodium DNA, nested PCR amplification of Pfcrt gene of Plasmodium falciparum Exon2 gene region and K13 domain of kelch and sequencing, sequencing and sequence 2655199 PF3D7_1343700 reference sequence were compared with MEGA 5.04, Exon2 Arlequin 3.01 Pfcrt gene analysis software, the kelch domain of K13 gene haplotype, haplotype between the expected heterozygosity (He) genetic index differentiation and between groups (Fst), making the intermediary network evolution haplotype map with Network 4.6.0 software, using Epi Da TA 3.1 software to establish database, SPSS 21 statistical software for data analysis. The results were collected blood samples of 234 cases of malignant malaria, Pfcrt Exon2 gene, K13 gene kelch domain of nested PCR amplification products and successfully sequenced 192 copies. Among them, Yunnan province local infection and 12 blood samples were collected from Africa, Burma cases were respectively. 30 and 150.218 Exon2 region of Pfcrt gene were DNA sequence had 7 haplotypes, He 0.2385 missense mutation sequences accounted for 83% (181/218), 72~76 loci showed single mutation (CVMNT), double mutant (SVMNT), three (CVIET) mutation rates were 1.8% (4/218). 6.4% (14/218) and 74.8% (163/218), 7 haplotypes with chloroquine sensitive CVMNK as starting, according to the single, K13 kelch gene DNA domain sequences and three double mutant.192 path gradually evolved blood samples of 21 haplotypes, He 0.0449 missense mutation sequence Accounted for 35.9% (69/192), in the 16446676 and 9 loci were only single mutation, the mutation rate of F446I is the highest, was 25% (48/192), intermediary network figure 21 haplotypes showed the "star shaped" layout. Yunnan province local P.falciparum populations between Burma population and the minimum is 0.020 Fst, 3.Pfcrt gene chloroquine sensitive CVMNK in blood, K13 gene kelch domain mutation ratio was 20.6% (7/34), chloroquine resistant type CVMNT detection ratio of CVIET in blood samples were 1/4 and 36.4% (51/140). Conclusion the Plasmodium falciparum epidemic reporting cases in Yunnan Province, the resistance gene of chloroquine, artemisinin related mutation rate was 27.1% there may be a population expansion mode, and between artemisinin resistance gene mutant.

【作者单位】：云南省寄生虫病防治所云南省疟疾研究中心云南省虫媒传染病防控研究重点实验室;大理大学基础学院;
【基金】：国家自然科学基金国际(地区)合作与交流项目(No.81220108019);国家自然科学基金地区科学基金项目(No.81660559) 云南省科技项目应用基础研究计划青年项目(No.Y0120150295)~~
【分类号】：R382.31
【正文快照】： kelch domain were amplified with nest-PCR.PCR products were sequenced,and the sequences were aligned with thereference sequences 2655199 and PF3D7_1343700.The haplotype number,expected heterozygosity(He),and geneticdifferentiation(fixation index,Fst)of t

【相似文献】